Among post-translational modifications, protein phosphorylation is particularly relevant to cancer biology and therapy. However, despite advances in proteomics, it is still difficult to pinpoint phosphorylation sites in proteins. The long-term goal of this project is to develop and commercialize a multiplexed method for isolating and identifying phosphorylation sites based on phosphorylation-specific antibodies. This method would contribute to the development of a new generation of drugs tailored to inhibit specific protein kinases with roles in cancer by identifying new phosphorylation sites that could become targets for cancer diagnosis and treatment. During Phase I we will develop an immunoaffinity method for multiplexed phosphopeptide sorting. We will address key issues qualitatively to establish the method and its tools. We will show that the method can isolate phosphopeptides from simple model systems and complex biological samples, evaluating results with MALDI-TOF mass spectrometry. We will show that isolated peptides can be further analyzed to identify sequences and phosphorylation sites using capillary LC-MS/MS, MS3, chemical modification, and enzymatic treatment as needed to generate informative spectra. Novel, ambiguous, or especially important phosphorylation sites will be confirmed by comparing the spectra of peptides synthesized to have the identified sequences and phosphorylation sites to the spectra of the isolated, natural phosphopeptides.